A central question in exercise physiology is how the balance between carbohydrate and fatty acid oxidation in skeletal muscle is altered to meet the metabolic demands of muscle contraction. We're using 13C NMR and non-steady-state isotopomer analysis to directly quantify the relative oxidation of two competing substrates in rat skeletal muscle. We found that [13C]acetate and [13C]lactate were taken up and oxidized by both resting and contracting skeletal muscle, and that high-intensity muscle contractions altered the pattern of substrate utilization such that the relative oxidation of acetate decreased while that of lactate remained unchanged or increased. However, because of the low sensitivity of 13C NMR, we had to pool two gastrocnemius muscles and four soleus muscles in order to acquire interpretable 13C NMR spectra. To overcome this limitation, we recently developed and validated a more sensitive method to analyze 13C enrichment in tissue extracts using 1H NMR. Using this new method, we have obtained data from individual rat gastrocnemius (~1.5 g wet weight) and soleus (~0.15 g wet weight) muscles. Glutamate C4 enrichment values obtained by 1H NMR were highly correlated with measurements obtained by 13C NMR non-steady-state isotopomer analysis. We are now applying these same techniques to examine muscle metabolism in mice. Our preliminary results indicate that 1H NMR is sensitive enough to detect glutamate C4 fractional enrichment in extracts from tissue samples as small as the mouse soleus muscle (10 mg wet weight). (Collaborative 5) REPORT PERIOD: (09/01/97-08/31/98)